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July 2017:
Congratulations to Yunyi Wang for receiving the JMR award at the International Society of Magnetic Resonance Conference

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Ligand Binding

We are exploiting dynamic nuclear polarization (DNP) for the development of new tools that enable the rapid characterization of dynamic chemical processes. In the life sciences, these tools enable the investigation of protein-ligand interactions with applications in drug discovery and implications for the fundamental understanding of the behavior of biological macromolecules.

Hyperpolarized NMR of Protein-Ligand Interactions for Drug Discovery
Illustration of polarization transfer in competitive ligand bindingUsing dissolution dynamic nuclear polarization (DNP), in a single scan we routinely achieve a signal gain of three to four orders of magnitude when compared to conventional NMR. The technique of hyperpolarization has opened up an alternative way to study protein-ligand interactions. It enables not only the direct detection of ligand binding but also kinetics of ligand interactions with protein. We have investigated competitive binding of two ligands to a protein by observing protein-mediated magnetization transfer between the two ligands. 1H spins of one of the two ligands are hyperpolarized and the fraction of this polarization induces NOE to the protein binding pocket which can be transferred to the second ligand. This effect is termed “hyperpolarized binding pocket NOE”. The enhanced signal intensities of the second ligand confirm that the two ligands competitively bind to a protein and also confirm the mode of binding of the second ligand due to the signal enhancement of specific protons. Moreover, relative signal build-up rates contain structural information on the binding epitope. Fluorine-NMR spectroscopy has become a powerful tool to explore protein-ligand interactions benefited from 100% natural abundance of 19F. When DNP technique is applied to 19F-NMR, submicromolar concentrations of fluorinated molecules are detectable which can benefit drug discovery. It is amenable to detect protein-ligand interactions in slow exchange which is limited by traditional NMR methods. We are developing techniques for the use of 19F-DNP to investigate ligand binding and binding dynamics. The dissociation constants for fluorinated ligands are calculated from a single one-dimensional spectrum which showed the ligand in free and bound states.

Hyperpolarized Reporters to Facilitate Screening
Illustration of an D-DNP experiment employing a reporter ligandScreening for ligand binding at physiological concentration can be challenging due to low signals. We are developing methods to rapidly determine the binding affinity of a ligand of interest by using a hyperpolarized reporter ligand. The reporter ligand contains a readily detectable functional group, for example including a fluorine atom, and becomes displaced from the binding site upon addition of the ligand of interest. This displacement is reflected in a readily detectable change in spin relaxation of the reporter. After establishing reference rates for the reporter, it becomes possible to measure the binding affinity of a ligand using a single, rapid hyperpolarized relaxation scan.

Binding Pocket Structure
Spectra arising from the transfer of polarization from a ligand to proteinProtein and bound ligand structure can be characterized using polarization transfer by intermolecular NOE. By mixing the protein with its hyperpolarized ligand, signals of protein protons, which are located close to the ligand, can be selectively enhanced. Using a 13C single quantum selection, observed NOE peaks in the 1D DNP 1H spectra become well-resolved. These observed peaks can then be used as structural constraints for the binding pocket.